The purpose of this study is to investigate 1) the effects of various dietary treatments on disease progression in the glycogen storage diseases (GSD), 2) the molecular basis and genotype-phenotype correlations of glycogen storage disease, 3) the mechanism and treatment of renal disease associated with type I GSD, and 4) development of recombinant enzyme replacement therapy for GSD type II. Methods: Patients with enzymatically confirmed diagnoses of glycogen storage disease are admitted to the Clinical Research Center. Laboratory evaluations include cardiac (EKG and echocardiogram), liver (ultrasound and blood chemistries) and muscle function (ischemic exercise test, EMG, nerve conduction and muscle strength). Peripheral blood is obtained from patient and parents for direct molecular analysis and lymphoblastoid cell transformation. The molecular findings are correlated with the type and severity of clinical phenotype expression of the disease. Results: In type IV glycogen storage disease, we have demonstrated that hepatic and neuromuscular forms of GSD-IV are caused by mutation in the same glycogen branching enzyme gene. We have also identified a mutant allele which has significant retention of branching enzyme activity. This mutant allele is only seen in patients with a non- progressive hepatic form of the disease and may be the reason for the mild disease. In type III glycogen storage disease we have identified the first pathogenic mutation and established the difference of subtype IIIa and IIIb at the molecular level. A striking association of mutations in exon 3 of the debranching enzyme gene also provides insight into mechanisms controlling tissue-specific expression of the gene. We are focusing on the development of recombinant enzyme replacement therapy for Pompe disease. Infantile Pompe disease is a fatal genetic muscular disorder caused by a deficiency of acid A-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid-glucosidase cDNA driven by the cytomegalovirus promoter. This was transfected into aminoglycoside phosphotransferase and dihydrofolate reductase-deficient Chinese hamster ovary cells. After selection with Geneticin and amplification with methotrexate, a cell line producing high levels of the A-glucosidase was established. The enzyme is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid A-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection high enzyme levels are seen in heart and liver in animals. An efficient production system now exists for recombinant human acid glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease. Significance: This study will provide us a better understanding of the long term complications and its pathogenesis in glycogen storage disease. Molecular study and genotype-phenotypes correlations will provide a non-invasive way of diagnosis of glycogen storage disease and the use of genotype to predict the clinical outcome. A high-level production system of recombinant human acid-glucosidase which targets muscle have potential for enzyme replacement therapy in Pompe disease (GSD type II). Future Plans: 1. Continue genotype-phenotype correlations and investigate molecular mechanisms in glycogen storage disease. 2. Scale-up human recombinant acid A-glucosidase product in a GMP facility for clinical trial.